Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome

Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific TRAP-seq and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1−/y). Our results identify a significant increase in the translation of ribosomal proteins (RPs) upon mGlu1/5 stimulation that coincides with a reduced translation of long mRNAs encoding synaptic proteins. These changes are mimicked and occluded in Fmr1−/y neurons. Inhibiting RP translation significantly impairs mGluR-LTD and prevents the length-dependent shift in the translating population. Together, these results suggest that pathological changes in FX result from a length-dependent alteration in the translating population that is supported by excessive RP translation.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Emily K Osterweil

4/27/2022
Proteomics data were collected using Spectronaut 13.7 Proteomics data were analysed using Limma R package 3.40.6 (Bioconductor). FDRs were computed with fdrtool R package version 1.2.16. Individual gene plots were created with DEP R package version 1.6.1. For RNA-seq, reads were mapped to the Mus musculus primary assembly (Ensembl release v88) using STAR 2.4.0i. Reads that were uniquely aligned to annotated genes were counted with featureCounts 1.4.6-p2. Differential expression analyses were performed using DESeq2_1.18.1 (Bioconductor). All GSEA and GSA were performed with Piano package version 1.18.1 (Bioconductor). Custom script used to calculate 5'UTR length is available at https:// github.com/sidbdri/empirical_utrs. Imaging analyses were performed using IMARIS 9.1.7 & 9.2.0. Desitometry of immunoblots was performed using ImageStudio Lite v5.0 and FIJI v1.51.
Additional data generated in this study is provided in the Supplementary Information and Source Data file. All renewable reagents and protocols will be available upon reasonable request.
Sample sizes for were chosen to minimize sample-to-sample variability as determined in previous studies (Thomson et al., 2017;Osterweil et al., 2013).
For molecular biology and biochemistry experiments, outlier analyses were performed however no data points were ultimately excluded. For LTD experiments unstable recordings (baseline drift +/-5%) were removed from analysis prior to unblinding. For the CX+DHPG TRAP-seq dataset, one sample was excluded based on poor quality read mapping.
Experimental samples were taken from multiple littermate pairs on several different experimental days. All results shown are averages of at least 3 independent experiments. With the exception of LTD recordings, each experiment contained yoked controls for genotype and treatment. The variability of effect sizes between yoked samples can be seen in the individual data points in each plot.
Littermate male mice were assigned to groups randomly and interleaving performed wherever possible.
All experiments and analyses were performed blind to genotype and treatment.
Validation statements for all antibodies used were supplied by the manufacturer for the species and specification. Information on this validation can be found on the manufacturer's website for each antibody listed.